Nomura Masaharu, Fukuda Tetsuya, Fujii Kiyonaga, Kawamura Takeshi, Tojo Hiromasa, Kihara Makoto, Bando Yasuhiko, Gazdar Adi F, Tsuboi Masahiro, Oshiro Hisashi, Nagao Toshitaka, Ohira Tatsuo, Ikeda Norihiko, Gotoh Noriko, Kato Harubumi, Marko-Varga Gyorgy, Nishimura Toshihide
Dept, of Surgery I, Tokyo Medical University, Tokyo, Japan.
J Clin Bioinforma. 2011 Sep 3;1(1):23. doi: 10.1186/2043-9113-1-23.
Large cell neuroendocrine carcinoma (LCNEC) of the lung, a subtype of large cell carcinoma (LCC), is characterized by neuroendocrine differentiation that small cell lung carcinoma (SCLC) shares. Pre-therapeutic histological distinction between LCNEC and SCLC has so far been problematic, leading to adverse clinical outcome. We started a project establishing protein targets characteristic of LCNEC with a proteomic method using formalin fixed paraffin-embedded (FFPE) tissues, which will help make diagnosis convincing.
Cancer cells were collected by laser microdissection from cancer foci in FFPE tissues of LCNEC (n = 4), SCLC (n = 5), and LCC (n = 5) with definite histological diagnosis. Proteins were extracted from the harvested sections, trypsin-digested, and subjected to HPLC/mass spectrometry. Proteins identified by database search were semi-quantified by spectral counting and statistically sorted by pair-wise G-statistics. The results were immunohistochemically verified using a total of 10 cases for each group to confirm proteomic results.
A total of 1981 proteins identified from the three cancer groups were subjected to pair-wise G-test under p < 0.05 and specificity of a protein's expression to LCNEC was checked using a 3D plot with the coordinates comprising G-statistic values for every two group comparisons. We identified four protein candidates preferentially expressed in LCNEC compared with SCLC with convincingly low p-values: aldehyde dehydrogenase 1 family member A1 (AL1A1) (p = 6.1 × 10-4), aldo-keto reductase family 1 members C1 (AK1C1) (p = 9.6x10-10) and C3 (AK1C3) (p = 3.9x10-10) and CD44 antigen (p = 0.021). These p-values were confirmed by non-parametric exact inference tests. Interestingly, all these candidates would belong to cancer stem cell markers. Immunohistochmistry supported proteomic results.
These results suggest that candidate biomarkers of LCNEC were related to cancer stem cells and this proteomic approach via FFPE samples was effective to detect them.
肺大细胞神经内分泌癌（LCNEC）是大细胞癌（LCC）的一种亚型，其特征是具有与小细胞肺癌（SCLC）相同的神经内分泌分化。迄今为止，LCNEC与SCLC之间的治疗前组织学区分一直存在问题，导致不良的临床结果。我们启动了一个项目，使用蛋白质组学方法，通过福尔马林固定石蜡包埋（FFPE）组织建立LCNEC特有的蛋白质靶点，这将有助于做出令人信服的诊断。
通过激光显微切割从具有明确组织学诊断的LCNEC（n = 4）、SCLC（n = 5）和LCC（n = 5）的FFPE组织中的癌灶收集癌细胞。从收获的切片中提取蛋白质，用胰蛋白酶消化，然后进行HPLC/质谱分析。通过数据库搜索鉴定的蛋白质通过光谱计数进行半定量，并通过成对G统计进行统计排序。使用每组10例病例进行免疫组织化学验证结果，以确认蛋白质组学结果。
从三个癌症组中鉴定出的总共1981种蛋白质在p < 0.05的条件下进行成对G检验，并使用包含每组两两比较的G统计值坐标的三维图检查蛋白质表达对LCNEC的特异性。我们鉴定出四种与SCLC相比在LCNEC中优先表达的蛋白质候选物，其p值令人信服地低：醛脱氢酶1家族成员A1（AL1A1）（p = 6.1 × 10-⁴）、醛糖酮还原酶家族1成员C1（AK1C1）（p = 9.6×10-¹⁰）和C3（AK1C3）（p = 3.9×10-¹⁰）以及CD44抗原（p = 0.021）。这些p值通过非参数精确推断检验得到证实。有趣的是，所有这些候选物都属于癌症干细胞标志物。免疫组织化学支持蛋白质组学结果。
这些结果表明，LCNEC的候选生物标志物与癌症干细胞有关，并且这种通过FFPE样本的蛋白质组学方法有效地检测到了它们。
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